Development of a new method of analysing chemotactic deactivation of humanneutrophil granulocytes

Measurement of chemotactic migration of human neutrophil granulocytes (PMN) induced by chemotaxins serves as a simple and reliable method for assessin g the expression of chemotaxin receptors. Incubation of PMN with a certain chemotaxin leads to a diminished chemotactic migration towards this chemota xin. This is called chemotactic deactivation. We developed a new deactivati on chamber to determine chemotaxis and chemotactic deactivation of human PM N. This novel chamber is a modification of the commercially available acryl ic 48-well microchemotaxis chamber consisting of an upper block with wells drilled all the way through the block and a blind-well lower block. Both bl ocks are separated by a polycarbonate membrane. PMN from the wells in the u pper block migrate through the pores of the membrane into the wells of the lower block containing the chemoattractants. Migrated PMN on the lower side of the PC membrane were quantified after staining by measuring specific li ght absorbance. The chemotactic activity is quantified as a ratio of stimul ated migration and random migration (chemotactic index = CI). For our novel chamber, only the upper blocks of this commercial chamber were connected l ike a sandwich, including a polyvinylpyrrolidone-free polycarbonate membran e with a pore size of 3 mum. The wells in the upper compartment were filled with 5 X 10(4) PMN and deactivating chemotaxin. The lower block was then f illed with the chemotactic stimulus and the chamber was then incubated in h umidified air with 5% CO2 atmosphere at 37 degreesC. The influence of cell concentration, incubation time, chemotactic factor concentration, pore size and alkaline treatment of polycarbonate membranes on migrational activity of PMN have been investigated. The technique was rigorously standardized in order to optimize the assay conditions. The method is relatively simple, s ensitive and fast. The determination of chemotaxis and deactivation are per formed in the same chamber, thus avoiding cell loss due to nonspecific adhe rence in other incubation tubes. The chamber can be used to characterize th e chemotactic activity of chemoattractants of unknown structure via known a nd unknown receptors. This new chamber can be very helpful in detecting unk nown chemotactic stimuli, which are not detectable by, for example, antibod ies. (C) 2001 Elsevier Science B.V. AII rights reserved.